Abstract

E. piscicida effector VgrG disturb iron homeostasis in fish cells. • VgrG induces NCOA4-mediated ferritinophagy for ferritin degradation. • VgrG facilitates the colocalization of ferritin with autophagosomes and lysosomes. • VgrG and inducible ferritinophagy enable bacteria to survive inside fish cells. • The deletion of vgrG gene reduces E. piscicida -caused mortality of zebrafish. Edwardsiella piscicida is an intracellular bacterial pathogen that causes intestinal injury and hemorrhagic sepsis in marine and freshwater animals. VgrG (valine-glycine repeat protein G) has been identified as a crucial virulence factor in the type VI secretion system (T6SS) of the bacterium, but its role and mechanism involved in E. piscicida- host interactions remain unclear. In this study, we found that the wild-type E. piscicida strain markedly induced host cell ferritin degradation and elevated intracellular Fe²⁺ levels, but a vgrG deletion mutant (Δ vgrG ) strain failed to induce similar effects in fish cells, indicating the role of VgrG in the regulation of iron metabolism. However, in the NCOA4 (nuclear receptor coactivator 4, a selective cargo receptor that binds ferritin)-knockout fish cells, VgrG did not alter ferritin protein expression and E. piscicida -promoted intracellular Fe²⁺ levels, suggesting that VgrG caused iron storage disorders via NCOA4-dependent ferritinophagy. In support of this notion, VgrG overexpression was found to facilitate the co-localization of ferritin with autophagosomes and lysosomes, and also drove the interaction of NCOA4 with lysosomes, strengthening the involvement of this effector in mediating NCOA4-dependent ferritinophagy. Notably, in NCOA4-knockout cells, the inhibitory effect of VgrG on the intracellular growth of E. piscicida was further magnified, particularly highlighting the role of VgrG-induced ferritinophagy in augmenting bacterial intracellular survival. Moreover, zebrafish infected with the Δ vgrG strain had a much h